The long-term goal of this research is to study the endothelial surface layer (ESL) expressed on vascular endothelium of microvessels in the context of its influence on microvascular hemodynamics in venules and arterioles and its role as a barrier to spontaneous rolling of leukocytes from the free stream in venules. The implications of the ESL across a broad range of fields in microvascular physiology have recently been established in work from our laboratories and in that of others. Results of this research will address basic physiologic function in the areas of microvascular flow resistance and inflammation and will thus have direct bearing on health and the human condition. Progress on advancing our understanding of the ESL and its role in microvascular physiology has been slow owing to experimental challenges in visualizing and interrogating the structure in vivo. However, recently we have developed and verified the accuracy of a comprehensive set of novel analytical tools that are capable of detecting the presence of the ESL and revealing its hydrodynamically relevant thickness in vivo. Using micro-particle image velocimetry data of blood flow in microvessels in vivo, and our new microviscometric method for accurately analyzing microvascular hemodyna.mics, we will (1) test whether the ESL increases resistance to blood flow in microvessels and quantitatively determine the Fahraeus and Fahraeus-Lindqvist effects in mouse skeletal-muscle venules in vivo, (2) test whether a hydrodynamically relevant ESL exists on arterioles in vivo, (3) test whether a physiologically typical ESL exists on the surface of a confluent monolayer of cultured endothelial cells, and (4) test whether the ESL acts as an anti-inflammatory barrier that prevents primary capture and subsequent rolling of leukocytes from the free stream in post-capillary venules in vivo. Our micro-viscometric method will be used extensively to interpret results and direct the experimental studies. With the new tools we have recently developed, we are poised to definitively resolve long-standing uncertainties in microvascular hemodynamics, conduct essential tests for the presence of the ESL on cultured endothelial-cell monolayers, and gain new insight into the potential barrier function of the ESL in inflammatory states.